DENV-2 3'UTR dumbbell structure is a critical factor for viral infection and dissemination in mosquito.

Unlabelled: Dengue virus (DENV) is a mosquito-borne flavivirus with the capability of replicating and spreading between vertebrate and invertebrate hosts, which poses a huge threat to global public health. Understanding the molecular mechanism of its replication and transmission in mosquitoes is crucial for controlling the dengue epidemic. The 3' untranslated region (UTR) has been identified as a mediator for viral replication, pathogenicity, and transmission, but its effect on DENV in the mosquito host remains unknown. This study investigated the role of the dumbbell (DB) structure within the 3'UTR of DENV-2 during mosquito infection. The results showed that the absence of the DB structure reduced DENV-2 viral replication efficiency in both mosquito cell lines and mosquito adults. The DB structure is essential for DENV-2 to escape from the midgut infection and scape barriers, but not for the salivary gland infection and escape barriers. The transcriptome analysis also revealed that the absence of the DB structure increased the expression of Defensin A and Defensin C, which can inhibit viral replication in midgut and spread to hemolymph at 7-10 days post-infection. Through siRNA-mediated knockdown and overexpression experiments in C6/36 cells, we further established that Defensin C exhibits particularly inhibitory effects on the absence of DB structure DENV-2. Our findings highlight the critical role of the DB structure in DENV-2 adaptation to the mosquito host and provide insights into the molecular mechanisms underlying flavivirus transmission, contributing to the understanding of flavivirus-host interactions and assisting the development of strategies for controlling dengue fever.

Importance: Dengue virus (DENV) transmitted by Aedes mosquitoes threatens global health. This study focuses on the dumbbell (DB) structure in DENV-2's 3' untranslated region, which is crucial for viral replication and transmission in mosquitoes. We discovered that the absence of the DB structure reduces DENV-2's replication efficiency and impairs its ability to evade the mosquito midgut barrier. Transcriptome data suggest that the DB structure may facilitate viral escape by modulating the expression of Defensin A and Defensin C. Functional validation in C6/36 cells demonstrates that Defensin C acts as an inhibitor, suppressing the replication of DENV-2 with the absence of DB structure and revealing a novel RNA structure-mediated immune evasion strategy. The findings of this study highlight the critical role of the DB structure in DENV-2 adaptation to mosquito hosts and provide valuable insights into the mechanisms of flavivirus transmission and potential strategies for dengue control.